Scc2 counteracts a Wapl-independent mechanism that releases cohesin from chromosomes during G1

Acknowledgements Maria Demidova conducted initial experiments that this study expanded on. We are grateful to Tomo Tanaka and Seiji Tanaka for supplying reagents. We thank all members of the Nasmyth group for valuable discussions, technical assistance and critical reading of the manuscript. This work was funded by the Wellcome Trust Senior Investigator Award, Grant Ref 107935/Z/15/Z and Cancer Research UK Programme Grant, Grant Ref 26747 to KN. BH is funded by (202062/Z/16/Z).Peer reviewedPublisher PD

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this ‘internal standard’ calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics

Transport of DNA within cohesin involves clamping on top of engaged heads by Scc2 and entrapment within the ring by Scc3.

In addition to extruding DNA loops, cohesin entraps within its SMC-kleisin ring (S-K) individual DNAs during G1 and sister DNAs during S-phase. All three activities require related hook-shaped proteins called Scc2 and Scc3. Using thiol-specific crosslinking we provide rigorous proof of entrapment activity in vitro. Scc2 alone promotes entrapment of DNAs in the E-S and E-K compartments, between ATP-bound engaged heads and the SMC hinge and associated kleisin, respectively. This does not require ATP hydrolysis nor is it accompanied by entrapment within S-K rings, which is a slower process requiring Scc3. Cryo-EM reveals that DNAs transported into E-S/E-K compartments are 'clamped' in a sub-compartment created by Scc2's association with engaged heads whose coiled coils are folded around their elbow. We suggest that clamping may be a recurrent feature of cohesin complexes active in loop extrusion and that this conformation precedes the S-K entrapment required for sister chromatid cohesion

The cohesin ring uses its hinge to organize DNA using non-topological as well as topological mechanisms

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis

Releasing activity disengages Cohesin’s Smc3/Scc1 interface in a process blocked by Acetylation

Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin’s Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin’s association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase- independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation

Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening

Cohesin stably holds together the sister chromatids from S phase until mitosis. To do so, cohesin must be protected against its cellular antagonist Wapl. Eco1 acetylates cohesin's Smc3 subunit, which locks together the sister DNAs. We used yeast genetics to dissect how Wapl drives cohesin from chromatin and identified mutants of cohesin that are impaired in ATPase activity but remarkably confer robust cohesion that bypasses the need for the cohesin protectors Eco1 in yeast and Sororin in human cells. We uncover a functional asymmetry within the heart of cohesin's highly conserved ABC-like ATPase machinery and find that both ATPase sites contribute to DNA loading, whereas DNA release is controlled specifically by one site. We propose that Smc3 acetylation locks cohesin rings around the sister chromatids by counteracting an activity associated with one of cohesin's two ATPase sites. Tight regulation of DNA entrapment and release by the cohesin complex is crucial for its multiple cellular functions. Elbatsh et al. find that cohesin's release from DNA requires an activity associated with one of its ATPase sites, whereas both sites control cohesin's loading onto DNA

Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes.

Cohesin's association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being 'clamped' by Scc2 and ATP-dependent engagement of cohesin's Smc1 and Smc3 head domains. Scc2's replacement by Pds5 abrogates cohesin's ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1's ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin's coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1's hinge that alters how Scc2 and Pds5 interact with Smc1's hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin's hinge, which in turn requires coiled coil folding

Probing the cohesin loading reaction using forward genetics and quantitative ChIP-sequencing

Cohesion between sister chromatids that are newly formed in S-phase is crucial to ensure the fidelity of chromosome segregation. The cohesin complex mediates cohesion and is comprised of two SMC proteins, Smc1 and Smc3, with a kleisin subunit, Scc1. The SMCs interact via their hinge domains to form a v-shaped heterodimer, with Scc1 forming a bridge between their head domains to produce a ring structure that topologically entraps sister chromatids. In order to confer cohesion, cohesin needs to be loaded onto DNA, a process that is poorly understood. It is known that cohesin loading depends on a loading complex comprised of Scc2 and Scc4, ATP hydrolysis and presumably opening of at least one of the interfaces of the cohesin ring. Using the recently developed technique, quantitative ChIP-seq, we show that Pds5, a cohesin-associated protein, is displaced at centromeres from the cohesin ring during the loading reaction. This event is accompanied by the engagement of SMC ATPase heads in the presence of ATP. Upon ATP hydrolysis, the loading reaction completes with DNA entrapment, translocation to the pericentromeric sequences and the displacement of Scc2 by Pds5. We also describe mutations in Scc2, Smc1 and histone proteins H2A and H2B that enable loading in the absence of Scc4. The mutations in Scc2 alter the dynamics of the association between the loading complex and cohesin, reducing dissociation during the translocation step. All of the mutations are able to restore loading along chromosome arms but not at centromeres where Scc4 has a particular role. However, the Smc1 mutation hinders loading at the centromere, even in the presence of Scc4, due to a defect in the second step of the loading reaction, namely translocation driven by ATP hydrolysis. We suggest that this discrepancy in functionality at the arms and centromere is due to changes in the hinge accompanying both the first and second steps. The rate limiting reaction is the first step at arms and the second at the centromeres. The histone mutants show that nucleosome occupancy plays an important role, at least on chromosome arms, but we find no evidence that this involves the RSC complex.</p

Probing the cohesin loading reaction using forward genetics and quantitative ChIP-sequencing

Cohesion between sister chromatids that are newly formed in S-phase is crucial to ensure the fidelity of chromosome segregation. The cohesin complex mediates cohesion and is comprised of two SMC proteins, Smc1 and Smc3, with a kleisin subunit, Scc1. The SMCs interact via their hinge domains to form a v-shaped heterodimer, with Scc1 forming a bridge between their head domains to produce a ring structure that topologically entraps sister chromatids. In order to confer cohesion, cohesin needs to be loaded onto DNA, a process that is poorly understood. It is known that cohesin loading depends on a loading complex comprised of Scc2 and Scc4, ATP hydrolysis and presumably opening of at least one of the interfaces of the cohesin ring. Using the recently developed technique, quantitative ChIP-seq, we show that Pds5, a cohesin-associated protein, is displaced at centromeres from the cohesin ring during the loading reaction. This event is accompanied by the engagement of SMC ATPase heads in the presence of ATP. Upon ATP hydrolysis, the loading reaction completes with DNA entrapment, translocation to the pericentromeric sequences and the displacement of Scc2 by Pds5. We also describe mutations in Scc2, Smc1 and histone proteins H2A and H2B that enable loading in the absence of Scc4. The mutations in Scc2 alter the dynamics of the association between the loading complex and cohesin, reducing dissociation during the translocation step. All of the mutations are able to restore loading along chromosome arms but not at centromeres where Scc4 has a particular role. However, the Smc1 mutation hinders loading at the centromere, even in the presence of Scc4, due to a defect in the second step of the loading reaction, namely translocation driven by ATP hydrolysis. We suggest that this discrepancy in functionality at the arms and centromere is due to changes in the hinge accompanying both the first and second steps. The rate limiting reaction is the first step at arms and the second at the centromeres. The histone mutants show that nucleosome occupancy plays an important role, at least on chromosome arms, but we find no evidence that this involves the RSC complex.</p

Crystal Structure of the Cohesin Gatekeeper Pds5 and in Complex with Kleisin Scc1

Sister chromatid cohesion is mediated by cohesin, whose Smc1, Smc3, and kleisin (Scc1) subunits form a ring structure that entraps sister DNAs. The ring is opened either by separase, which cleaves Scc1 during anaphase, or by a releasing activity involving Wapl, Scc3, and Pds5, which bind to Scc1 and open its interface with Smc3. We present crystal structures of Pds5 from the yeast L. thermotolerans in the presence and absence of the conserved Scc1 region that interacts with Pds5. Scc1 binds along the spine of the Pds5 HEAT repeat fold and is wedged between the spine and C-terminal hook of Pds5. We have isolated mutants that confirm the observed binding mode of Scc1 and verified their effect on cohesin by immunoprecipitation and calibrated ChIP-seq. The Pds5 structure also reveals architectural similarities to Scc3, the other large HEAT repeat protein of cohesin and, most likely, Scc2